Methods of use of uncoated gel particles

ABSTRACT

The invention covers a method of implanting a living donor cell into a host animal without inflammatory response or rejection of the donor cell by the host animal, by obtaining an uncoated particle of a biocompatible, temperature-independent gel that encapsulates the living donor cell, wherein the uncoated particle provides a molecular weight cutoff that prevents host animal immune cells from entering the particle, yet does not have to prevent entry of host animal IgG and complement into the particle, and implanting the uncoated particle into the host animal.

BACKGROUND OF THE INVENTION

The invention relates to methods of use of gel particles such as beadsand spheres.

Gel microcapsules, e.g., of alginate, that contain a relatively smallnumber of living cells have been used to transplant donor cells intohost animals in both allografts, i.e., same-species transplants, andxenografts, i.e., different-species transplants. The microcapsules areused primarily in an attempt to immunoisolate the donor cells from thehost's immune system. In the case of alginate microcapsules, theyinclude an inner gel core and an outer semipermeable membrane or othercoating with a controlled porosity to prevent components of the host'simmune system from entering and destroying the cells within themicrocapsule cores.

Several methods for microencapsulating cells, e.g., pancreatic isletcells, in alginate gels have been investigated. These include thealginate-polylysine technique described in Lim et al., U.S. Pat. No.4,391,909 and Soon-Shiong et al., Transplantation, 54:769-774 (1992),the alginate-chitosan system described in Rha et al., U.S. Pat. No.4,744,933, and the polyacrylate encapsulation method described inSefton, U.S. Pat. No. 4,353,888. Each of these methods results inalginate gel microcapsules with an outer coating that is distinct fromthe inner core.

The alginate-polylysine technique, involves extruding a mixture of cellsand sodium alginate into a CaCl₂ solution using a droplet generationdevice to form temporary gelled droplets. These droplets are then coatedwith positively charged polylysine to form a semipermeable outermembrane or coating around the gelled droplets. Tests have shown thatthese microcapsules are unstable and produce an inflammatory andfibrotic response when implanted into the peritoneal cavity of animals.However, the addition of a third outer alginate layer over thepolylysine membrane has improved the biocompatibility of themicrocapsules, resulting in an increase in the duration of isletallograft function in diabetic rodents to more than a year, as describedin O'Shea et al., Biochem. Biophys. Acta, 804:133-136 (1984).

Although the alginate-polylysine microcapsules have been shown toprolong the survival of cells in allografts and xenografts, thesemicrocapsules have typically required adjunctive treatment withimmunosuppressive agents such as cyclosporin ("CsA"). However, when usedin therapeutic, i.e., immunosuppressant, dosages, these agents cause ahost of serious side effects including infection, cancer, and renaltoxicity. Thus, the use of immunosuppressive agents in therapeuticdosages is undesirable.

Nevertheless, immunosuppressive agents are still used. For example,Soon-Shiong et al., Transplantation, 54:769-774 (1992) and Soon-Shionget al., P.N.A.S., USA, 90:5843-5847 (1993), describe the use ofalginate-polylysine-alginate microcapsules for allografts of canineislets into diabetic dogs, both with continuous or temporary, e.g., 30day, immunosuppression with CsA. Both sets of dogs remained independentof insulin for an average of over 100 days.

In another report, Soon-Shiong et al., First Int'l Cong. Xenotrans., p.22 (Minneapolis, MN 1991), describes the prolongation of discordantislet xenograft function in streptozotocin-induced diabetic rats byalginate-polylysine microencapsulation. Microencapsulated canine andhuman islets were implanted intraperitoneally in the rats and comparedto nonencapsulated islet implants. Low dose CsA therapy was institutedin both groups for the duration of the study. Euglycemia was maintainedfor 43 to 123 days for canine islets, and 42 to 136 days for humanislets. In contrast, nonencapsulated islets achieved euglycemia for lessthan 2 days.

However, there are a few reports of uses of microcapsules withoutimmunosuppression. For example, Weber et al., Transplantation,49:396-404 (1990), describes a discordant, e.g., from unrelated species,xenograft in which alginate-polylysine microcapsules containing canineislets functioned for an average of only 11.5˜3 days in diabetic NODmice. However, immunosuppressive treatment with anti-CD4 monoclonalantibody allowed the cells in some of the recipient mice to remainfunctional for an average of 83 days.

In addition, Iwata et al., Diabetes, 38 (Supp. 1):224-25 (1988),describes the use of pancreatic islet cells encapsulated in agarose gelmicrospheres in concordant xenografts, i.e., transplants from different,but closely related species, e.g., rodent-to-rodent transplants, such ashamster cells into mice. No immunosuppressive agent was used in thisstudy, and the two mice remained normoglycemic for 29 and 53 days,respectively.

In a second similar concordant xenograft study, Iwata et al.,Transplantation Proc., 24:952 (1992), the immunosuppressive effect ofthe drug 15-deoxyspergualin on host mice was compared with control micethat received no immunosuppression. Iwata et al. concluded that theagarose microspheres without immunosuppression could not effectivelyprotect the concordant xenografts from rejection, because blood glucoselevels indicated that only 2 of 8 xenografts survived over 100 days.However, blood glucose levels indicated that 3 of 5 xenografts survivedover 100 days in mice receiving the immunosuppressive drug for 120 days(2.5 mg/kg/day) or 40 days (5.0 mg/kg/day).

In another study, Iwata et al., Transplantation proc., 24:934 (1992),used mouse islet allografts in agarose microspheres to achievenormoglycemia in diabetic mice without immunosuppression. Blood glucoselevels indicated that the majority of these allografts survived over 100days.

SUMMARY OF THE INVENTION

The present methods of using biocompatible, temperature-independent gelparticles, e.g., beads, are based on the discovery that donor cells,e.g., porcine, bovine, or canine islet cells, encapsulated in alginatebeads can be successfully transplanted into a host animal, e.g., mouse,rat, or dog, without any protective coating or semipermeable membranearound the beads, and with the use of only minimal doses, if any, ofimmunosuppressive or anti-inflammatory drugs. These simple, uncoatedbeads can be implanted into the host and provide effectiveimmunoisolation of the encapsulated cells without eliciting a fibroticresponse or a host immune rejection of the donor cells within the beads.

In general, the invention features a method of implanting a living donorcell into a host animal without inflammatory response or rejection ofthe donor cell by the host animal by obtaining an uncoated particleconsisting essentially of a biocompatible, temperature-independent gelthat encapsulates the living donor cell, wherein the uncoated particleprovides a molecular weight cutoff that prevents host animal immunecells from entering the particle, and does not prevent entry of hostanimal IgG and complement into the particle, and implanting the uncoatedparticle into the host animal.

As used herein, a "temperature-independent gel" is a gel that can begelled or crosslinked, e.g., by the addition of ions such as calcium,potassium, or barium ions, without a change in temperature. An "uncoatedparticle" refers to a bead, sphere, or other gel structure, e.g., acylinder, that is composed of a biocompatible, temperature-independentgel matrix without any surface or intermediate layer, e.g., in the formof a semipermeable membrane, of a permeability and molecular weightcutoff different from that of the gel matrix itself.

As used herein, "molecular weight cutoff" refers to the size of thelargest molecule that is not substantially blocked by a semipermeablemembrane surrounding a microcapsule or by the gel matrix itself in anuncoated gel particle, e.g., bead, according to the invention. Moleculeswith a molecular weight above the cutoff are substantially preventedfrom entering or leaving the microcapsule or gel particle. The coatingsof prior art alginate microcapsules generally provide a molecular weightcutoff of greater than 50,000 and less than 100,000 daltons. Theuncoated gel particles of the invention have a molecular weight cutoffof greater than about 500,000 daltons, i.e., molecules like IgG andcomplement can enter these gel particles, but host cells such asimmunocytes are prevented from entering these gel particles. Inaddition, this high molecular weight cutoff allows molecules secreted bythe encapsulated cells, e.g., Factor VIII or hormones, to exit the gelparticles.

The invention also features a method of implanting a living donor cellinto a host animal without inflammatory response or rejection of thedonor cell by the host animal by suspending the living donor cell in aliquid medium, the medium consisting essentially of water and abiocompatible, temperature-independent liquid gel, forming a droplet ofthe liquid medium that contains at least one living cell, solidifyingthe droplet to form a gel particle that encapsulates the living cell,whereby no outer coating is formed on the particle, and wherein theuncoated particle provides a molecular weight cutoff that prevents hostanimal immune cells from entering the particle, and does not prevententry of host animal IgG and complement into the particle, andimplanting the uncoated particle into the host.

In particular embodiments, when the liquid medium contains pancreaticislets, they can be present at a density of about 2 to 60 islets permm³, and more preferably at a density of about 10 to 35 islets per mm³i.e 10,000 to 35,000 islets per milliliter of the medium. When theliquid medium contains other living cells, they can be present at adensity of about 10⁵ to 10⁸ cells, and preferably 10⁶ to 10⁷ cells, permilliliter of the medium. The density depends on the size and metabolismof the individual islets.

Furthermore, the invention features a method of treating a disease in apatient caused by a deficient production of a substance in the patientby obtaining an uncoated particle consisting essentially of abiocompatible, temperature-independent gel that encapsulates a livingdonor cell that secretes the substance, wherein the uncoated particleprovides a molecular weight cutoff that prevents patient immune cellsfrom entering the particle, and does not prevent entry of patient IgGand complement into the particle, and implanting the uncoated particleinto the patient in a location and in a manner that allows the livingcell to remain physiologically active and secrete the substance into thepatient to treat the disease. For example, the uncoated particles can beimplanted into an immunoprivileged site in the patient.

In particular embodiments, the disease is diabetes and the donor cell isa pancreatic islet cell. The donor cell can be selected to secreteFactor IX, Factor VIII, an interleukin, an interferon, an endocrinehormone, a nerve growth factor, tumor necrosis factor alpha, aneurotropic factor, or a neurotransmitter. The disease can be diabetesmellitus, hepatic disease, amyotrophic lateral sclerosis, hemophilia,hypothyroidism, Parkinson's disease, acquired immune deficiencysyndrome, Duchenne's muscular dystrophy, infertility, epilepsy,Huntington's disease, hypoparathyroidism, a mood disorder, a motorneuron disease, osteoporosis, or Alzheimer's disease.

The invention also features an in vivo method of culturing a living cellby encapsulating the living cell in an uncoated particle consistingessentially of a biocompatible, temperature-independent gel, insertingthe uncoated particle into an animal, and allowing the animal to thrive,thereby culturing the cell.

The invention further features, an in vitro method of culturing a livingcell by encapsulating the living cell in an uncoated particle consistingessentially of a biocompatible, temperature-independent gel, placing theuncoated particle into a medium including nutrients and oxygen, andmaintaining a sufficient amount of nutrients and oxygen in the medium toallow the cell to thrive, thereby culturing the cell.

In addition, the invention features a method of manufacturing uncoated,temperature-independent gel particles containing living cells consistingof the steps of suspending the living cells in a liquid medium, themedium consisting essentially of water and a biocompatible,temperature-independent, liquid gel, forming a droplet of the liquidmedium, solidifying the droplet to form a gel particle that encapsulatesthe living cells, whereby no outer coating is formed on the particle,and storing the gelled uncoated particles in a nutrient medium tomaintain the viability of the living cells.

In all of these methods, the living donor cell can be obtained from aspecies that is the same as or different from the host animal, and canbe a genetically altered human cell. The host animal can be a dog or ahuman. The donor cell can be a porcine, bovine, canine, bacterial,fungal, or plant cell. In particular, the donor cell can be a pancreaticislet cell, or can secrete Factor IX, Factor VIII, an interleukin, aninterferon, an endocrine hormone, a nerve growth factor, tumor necrosisfactor alpha, a neurotropic factor, or a neurotransmitter.

In particular embodiments, the gel particle is spherical and has adiameter of from 50 to 6000 microns, and preferably from 2000 to 4500microns. The gel can be an alginate or alginate derivative, and thealginate can be crosslinked with an ion, such as the calcium in acalcium salt. The uncoated gel particle can be biodegradable, and therate of degradation of the gel in the uncoated particle can be selectedto match the life expectancy of the donor cell.

In other embodiments, the uncoated particle encapsulates an autologouserythrocyte in addition to the donor cell, or can be treated with anitric oxide inhibitor prior to implantation. In addition, the methodcan include the step of administering a drug to the host animal at adosage effective to inhibit fibrosis and inflammation of the uncoatedparticle, but at a dosage lower than that required to achieveimmunosuppression when the donor cell is implanted into the host animalwithout encapsulation. For example, the drug can be cyclosporin A and isadministered at a dosage that achieves a whole blood trough level ofless than about 100 ng/ml in the host animal. In addition, the drug canbe administered for up to several weeks, e.g., one month, or longer,after implantation, and is then no longer administered.

The invention also provides a variety of other features which enhanceimplant function and longevity including size of the beads, types of gelmatrices, and for islet cells, optimum cell densities. It is essentialthat the gel matrices keep host cells, i.e., immunocytes, physicallyseparated from the donor tissue cells. It is also important that theparticles have a sufficient size to keep antigens secreted by theencapsulated living cells from building up in the matrix and coming intodirect contact with the host, and to protect the encapsulated donorcells from small soluble or cytotoxic factors in the host such as nitricoxide, lymphokines, cytokines, and natural killer (NK) cytotoxicfactors. The charge and chemical properties of the matrix are alsoimportant in this respect.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of a mouse macrophage digesting a 18 dayold, fibrosed alginate gel bead (fibers in cell).

FIG. 2 is a graph showing the effect of porcine islet implants on plasmaglucose level in mice.

FIG. 3 is a graph showing the effect of bovine islet implants on plasmaglucose level in mice.

FIGS. 4 is a graph showing the effect of bovine islet implants on plasmaglucose level in rats.

FIGS. 5A and 5B are graphs showing the blood glucose Level in diabeticpatient dogs before and after implantation of canine islets with lowdose cyclosporin.

DETAILED DESCRIPTION

Various types of donor cells can be isolated, encapsulated, and thenimplanted into a host according to the present invention.

Isolation of Cells

Cells are isolated from surrounding tissues or grown in culture byprocedures known to the art, and are then suspended in a liquid mediumprior to encapsulation. For example, pancreatic islet cells wereprepared from either adult mongrel dogs, pigs, or bovine calves (0-2weeks old) by a modification of the methods of Warnock and Rajotte,Diabetes, 37:467 (1988), as previously described in Lanza et al.,P.N.A.S. USA, 88:11100-11104 (1991).

Briefly, aseptic, viable porcine pancreata were obtained under asepticoperating room procedures. After resection (warm ischemia for less thanabout 15 minutes), the glands were cannulated and infused with cold (4°C.) University of Wisconsin (UW) organ preservation solution. Pancreatictissues were dissociated using an intraductal collagenase digestionprocedure. The collagenase is delivered by peristaltic pump, and thedigested pancreas is mechanically disrupted in a polypropylenedissociation chamber containing 2-6 mm glass beads. The islets wereseparated from the exocrine tissue by discontinuous density gradientcentrifugation (27%, 20.5%, and 11% (w/v) FICOLL® (Sigma, F 9378) inEurocollins solution).

Isolated islets were then cultured for one day either in M199/Earle'smedium supplemented with 10% (vol/vol) fetal bovine serum, 30 mMHEPES,100 mg/dl glucose, and 400 IU/ml penicillin (canine), or in α-MEMplus10% heat-inactivated horse serum (bovine and porcine) in a humidifiedatmosphere of 5% CO₂ /95% air at 37° C. A typical yield of islets shouldbe in the range of 0.5-1.8×10⁶ islets for adult pancreas (400 gmwetweight, islet diameter 80-125 μm, purity 85-95%, viability greater than90% (see below). The cells may also be isolated by other procedures andcultured under other suitable conditions.

Ischemic deterioration of the islet cells is minimized by using tissuefragments of a suitable size, e.g., islet fragments should be less thanabout 120 microns, and preferably 40 to 100 microns, in diameter.Viability, growth, longevity, and/or function of the islet cells can beenhanced by co-culturing, i.e., by mixing other cell types in the liquidmedium prior to encapsulation. Useful cell types include cells whichsecrete growth hormone, e.g., GH-3 cells, or cells which secreteconnective tissue and/or extracellular matrix components, e.g.,fibroblasts and endothelial cells. In addition, cells, e.g., islets, canbe co-cultured with red blood cells, hemoglobin, or other oxygencarrying agents to enhance oxygen availability. Islet quality controlprocedures are used to enable comparison of different lots of isletsprepared at different times. Purity (amount of islet tissue compared toexocrine tissue contamination) depends on the relatively uniquecharacteristic of pancreatic islets to rapidly take up diphenylthiocarbazone (dithizone). Islets are therefore incubated for five toten minutes with 50 μg/ml of dithizone (D5130, Sigma) to stain them red.The preparation is then examined under light microscopy for aqualitative estimate of purity. Quantification of purity is effected byislet dispersion and counting of stained and unstained cells, or with aspectrophotometric assay of dithizone uptake/μg DNA.

Viability can be determined by any one of several assays that depend onthe capability of viable cells to exclude certain dyes. For example, oneassay uses a combination of the fluorescent stains acridine orange,which stains only viable cells green, and propidium iodide, which stainsonly the nuclei of dead cells red. The islets are incubated with thedyes (acridine orange, Sigma A6014, 50 μg/ml, and propidium iodide,Sigma P4170, 2.5 μg/ml) in a PBS solution for 10 to 15 minutes and thendispersed into single cells. Counts of red and green fluorescing cellsare used to calculate % viability.

Insulin secretory activity of the islets is determined both in staticculture, e.g., expressed as units of insulin per islet volume, and basedon the capability of the islets to respond to graded concentrations ofglucose. These values are quantitatively established by measuring theinsulin secreted by islets exposed to a range of glucose concentrationsextending from 2.8 to 28 mM glucose.

Encapsulation

Once the cells are isolated and suspended in liquid medium, they must beencapsulated by a supporting gel matrix. Beads suitable for implantationinto a host animal include a number of living donor cells in a gelmatrix without any protective coating. Using standard techniques, a gelmatrix is formed by adding cells, e.g., pancreatic islets, to a solutionof nutrient medium and liquified gel, e.g., sodium alginate, to form asuspension, and then crosslinking the gel, e.g., by adding acrosslinking agent such as calcium chloride. The gel matrix can be anyone or a combination of a variety of substances that are biocompatiblewith the host animal, and are capable of maintaining cellular viabilityand physically supporting the tissue or cells in suspension, as long asthey have the required concentration and purity.

The gels must be temperature-independent, in that they can be gelled orcrosslinked, e.g., by the addition of ions such as calcium, potassium,or barium ions, without a change in temperature, which could be harmfulor fatal to the living cells to be encapsulated. Temperature-independentgels include alginates, carrageenans, and gums such as xanthan gum. Asused herein, the term alginate includes alginate derivatives. These gelsshould be treated to remove polyphenols, lipopolysaccharides,endotoxins, and other impurities using standard techniques.

Alginate is composed of blocks of 1,4 linked β-D-mannuronic acid (M) andα-1-guluronic acid (G) linked together, e.g., in alternating MG blocks.The preferred alginate is one formulated with a high G block content,e.g., at least about 60 percent. The higher the percentage of G blocks,the greater the pore size and the strength of the gel matrix. Inaddition, it has been noted that alginate gels with a high M blockcontent appear to be more immunogenic than gels with a high G blockcontent. See, e.g., Soon-Shiong et al., Transplant. Proc., 23:758-759(1991), and Soon-Shiong et al., Transplantation, 54:769-774 (1992).

The gel matrix should be sufficiently viscous to maintain the cells in adispersed state. When alginate is used as the gel matrix, it is added upto about 3%, preferably to about 1 to 2%, of the liquid medium, and thesolution is cross-linked to form a semisolid gel in which the cells aresuspended. These percentages provide a matrix that maintains its shapeand has sufficient mechanical strength to remain intact in vivo forseveral months.

For example, pancreatic islets can be encapsulated as follows. Afterpreculturing overnight, islet cells were suspended uniformly at adensity of 20,000 islets/ml, which is 20 islets/mm³, in a solution of1.5% (wt/vol) Pronova LVG sodium alginate (Protan, Drammen, Norway) inculture medium plus additives (α-MEM, 10 mMHEPES pH 7.1, penicillin, 2mM glutamine for porcine islets; and M199 with the same additives forcanine islets). A syringe pump was used to pump the suspension throughan air jet apparatus (containing a straight-edged 22 gauge needle) at aspeed of 3 ml/min. Droplets formed at the tip of the needle werestripped off by means of a concentric flow of air at an air speed of 7to 8 m/sec. The resulting droplets fell a distance of 4 cm and werecollected in a solution of 1.5% CaCl₂ in 10 mMHEPES (pH 7.1) to formgelled beads. These beads can be made in various sizes ranging fromabout 700 μm to 3500 μm in diameter by altering the air flow speed, thefaster the flow rate the smaller the beads.

Each bead contains approximately 1 to 25 islets. After three minutes,the beads were washed three times with culture medium (appropriate forthe species of islets in use), and were then cultured in a tissueculture incubator at 37° C. and 5% CO₂ until they were implanted.

Larger beads up to 3500 to 6000 mm in diameter, were or can be made in asimilar manner, or can be extruded through a syringe with a 14 gaugecatheter. Beads can also be made by other standard techniques, as longas the resulting beads have the preferred characteristics describedbelow.

The beads were cultured in vitro for up to four weeks, and the insulinsecretion compared to free islets, prepared as described above. Theinsulin secretory response of the beads was approximately 50 to 80% ofthat of the free islets. Histological examination at four weeks revealedviable endocrine tissue within the beads. The islets weremorphologically intact, and contained well granulated β-cells.

Specific Parameters for Uncoated Particles

The particles, e.g., beads, are preferably spherical in configurationand have a diameter of about 600 to 6000 μm, preferably 1500 to 3500 μm.Particles as small as 50 μm can be made. The preferred size is based ondiffusion distances from the surface of the bead to the cells within. Inaddition, smaller beads, e.g., 700 to 900 μm are suitable forallographic transplants, whereas larger beads, e.g., 2000 to 5000 μm,are preferred for xenographic transplants.

A spherical shape is preferred for the beads to present a smooth outersurface without edges, which tends to inhibit fibrotic encapsulation ofthe beads. The beads may be designed to be more or less biodegradabledepending on the intended use. For example, if the beads are intended tobreak down within a certain period of time, materials such as celluloseor collagen can be added to the gel matrix to facilitate the breakdown.However, as shown in FIG. 1, applicants have discovered that even purealginate beads are attacked and digested by macrophages after the gelparticle has been coated by fibrotic tissue by the host. FIG. 1 shows amacrophage with alginte fibers in the center of the cell. Otherdegradative mechanisms also occur. The resulting breakdown products areresorbed by the body, or excreted in the urine as segments ofcrosslinked or uncrosslinked alginate molecules. This breakdown of thebeads may start within a few weeks or months, or within a year, and iscontrolled by the size of the beads, the crosslinking agent used to formthe beads, and the added ingredients such as collagen, which dissolveson its own to weaken the bead structure after a few weeks. In general,this breakdown typically occurs after 6 to 12 months.

Other characteristics of the uncoated beads include (1) morphologicaland chemical properties, e.g., the smoothness of surface, the structureof the matrix, and the ability to react with other chemical substances,and (2) transport properties, e.g., permeability to microsolutes,nutrients, O₂, wastes, macrosolutes (e.g., insulin), essential proteins,and molecular weight cutoff to prevent immune cells(lymphocytes/macrophages) from entering the bead as discussed above.Both the morphological and transport characteristics are achieved by thegel matrix which physically isolates the donor cells from the host cellsand allows nutrients and oxygen to flow freely into the matrix, whichfacilitates viability of cells. In addition, the negative charge of somegels, e.g., sodium alginate, should aid in preventing proteins of thehumoral immune response (complement/cytokines) from entering the gelparticles.

Donor Cell and Host Characteristics

The living donor cells are preferably mammalian cells, but can also bebacterial, fungal, or plant cells that express or secrete a desiredprotein, hormone, or other substance. The characteristics of theseencapsulated donor cells are important to the survival of cells in theparticles once implanted into a host. For example, the total antigenicload should be kept as low as possible while still implanting asufficient number of donor cells to achieve the desired therapeuticeffect.

This antigenic load can be controlled by adjusting the density of cellsper bead and/or by adjusting the total number of beads implanted intothe host. These numbers vary depending on the cell type and the type ofhost. For example, in a dog example described below, diabetes wastreated with beads made from 32.0 ml of gel containing porcine islets ata density of about 20 per mm3. To standardize islet dosages, the EIN(equivalent islet number) can be used. This number is based on the isletvolume of a standard islet of 150 microns in diameter.

The total EIN implanted into a patient depends on the insulinrequirements of the patient, and on the metabolism, type, and quality ofthe islets, which is determined by in vitro tests of the encapsulatedislets prior to implantation as described herein. For example, it isknown that porcine islets produce more insulin than bovine or canineislets. The amount of insulin (insulin units) required by a patient isdetermined empirically on an individual basis, and is based on sugarlevels monitored several times per day. For example, diabetic dogs mayrequire about 5 to 40 units of insulin per day, whereas a typical humandiabetic patient may require 20 to 50 units per day. In all cases, theseamounts depend on the severity of the disease, diet, exercise, and otherfactors. About 1.0 to 2.5 million porcine islets are required to achievethis level of insulin production for a human patient.

In addition, the immunogenicity of the donor cells must be considered.For example, it is believed that fetal or neonatal tissue will provokeless of a host reaction than adult tissue. The donor tissue can also bemodulated to reduce its immunogenicity prior to implantation, e.g., byorgan culture, UV irradiation, and/or pretreatment with antibodies tomask the antigens on the surface of the donor cells. Organ culturingselectively removes dendritic cells (antigen presenting cells) from thedonor tissue since they die faster than other cells in culture. Cultureconditions such as high oxygen and low temperature are effective toselectively destroy the more sensitive dendritic cells. All of thesemethods of modulating donor tissues are described in Chapters 9, 10, 11of Lanza et al. (eds.), Immunomodulation of Pancreatic Islets (RGLandes, Tex., 1994), which is incorporated herein by reference.

The immune system of the host can also be modulated prior to or afterimplantation of the encapsulated donor cells to ensure survival of theimplanted cells. Allografts in mammals larger than mice or rats requirea short course of an immunosuppressant or anti-inflammatory drug at alow dosage. Discordant xenografts with small beads, e.g., 700 to 900 μmin diameter, in larger mammals such as humans also require adjunctiveimmunosuppression. As discussed below, minimal or no immunosuppressionor anti-inflammatory therapy is necessary with larger beads.

Immunosuppressant drugs include cyclosporine A ("CsA"), FK-506, anddeoxyspergualin. Anti-inflammatory/anti-fibrosis drugs include steroidaldrugs such as prednisone, and non-steroidal drugs such as ibuprofen andaspirin. Certain immunosuppressants such as CsA have an anti-fibrosiseffect at very low, "subtherapeutic" doses, e.g., at a so-called "wholeblood trough level" of less than 100 ng/ml when analyzed by HPLC.Initial doses can be higher, e.g., up to a few hundred ng/ml, withoutattaining a therapeutic, e.g., immunosuppressive, dose, which is in therange of 550 to 900 ng/ml in dogs for unencapsulated xenogeneic islets.Thus, CsA can be used as an effective anti-fibrotic without the need forany other drugs. The maintenance blood levels of less than 100 ng/ml,e.g., 30 ng/ml, in allografts can be discontinued within several weeksto less than three months.

In human patients, the maximum therapeutic dosage of CsA should be lessthan 800 ng/ml to avoid toxicity problems. However, according to theinvention, only low doses, e.g., an initial dosage of animmunosuppressive/anti-fibrotic agent of a few hundred ng/ml, and then amaintenance dosage of less than 100 ng/ml, should be administered.

In addition, according to in vitro observations, the living donor cellswithin the gel beads can be protected from cytotoxic nitric oxideradicals, by co-encapsulating the cells with autologous erythrocytes,which scavenge nitric oxide that may enter the gel beads once implanted,e.g., as described in Wiegand et al., Transplantation, 56:1206-1212(November 1993). In addition, the gel beads can be treated with nitricoxide inhibitors such as N^(G) -methyl-L-arginine prior to implantationto provide a protective effect.

Implantation

The beads can be simply implanted into a host by injection with astandard catheter or syringe, e.g., with a 16 gauge needle for beadsless than 1000 μm in diameter. Larger beads can be inserted via a smallincision, e.g., with a catheter or funnel-like device. The beads arepreferably implanted into the host intraperitoneally. The beads can alsobe implanted intramuscularly or subcutaneously. Alternatively, the beadscan also be implanted into immunoprivileged sites such as the brain,testes, or thymus, where the host's immune response is least vigorous,as described in Chapter 7 of Lanza et al. (eds.), Immunomodulation ofPancreatic Islets (RG Landes, Texas, 1994). In addition, the beads canbe inserted through a small surgically created opening using agun/trocar type device that slips the beads under the skin.

EXAMPLES Implantation of Porcine Pancreatic Islets into Mice and Rats

To determine whether encapsulated pancreatic islet cells can function,e.g., secrete insulin in a host animal, over extended periods of time,800±100 μm diameter beads, containing a total of between 10K and 100Kislets per animal, were implanted as discordant xenografts into mouseand rat diabetes mellitus models. Adult male Lewis rats (Charles River,Wilmington, MA) weighing 250 to 300 g, and C57BL/6J mice weighing about20 to 30 g were used as implant hosts. Diabetes mellitus was induced inthese animals by a single injection of streptozotocin ("STZ") ten tofourteen days prior to implantation of the beads. Rats were injectedwith 42 mg/kg body weight of STZ into the tail vein. Mice were injectedwith 165 mg/kg body weight of STZ into the peritoneal cavity.

Fasting plasma glucose concentrations (mg per dl) were measured by tailbleedings from both animals using a glucose oxidase method (BeckmanGlucose Analyzer 2, Fullerton, CA). Determinations were performed thriceweekly for one month, and then weekly for the duration of each study.Failure of the encapsulated islets to reverse hyperglycemia wasconsidered to have occurred when glucose concentrations exceeded 250 mgper dl on two consecutive testings.

Host animals were anesthetized with ketamine/xylazine (rats, 0.5 μl/gi.m.; mice, 5.0-7.5 μl/g i.p.) prior to implantation. The porcine isletswere isolated and encapsulated in 800 μm diameter beads as describedabove. Between 10K and 100K islets were implanted into the peritonealcavity of the rats or mice either with a 16 gauge catheter or through asmall (1-2 cm) midline incision. This corresponds to a total of about0.5 to 5.0 mls of gel which is formed into the beads, i.e., the isletsare present in the gel at a density of about 20K islets/ml of gel. Thewound was closed in two layers with 4-0 silk suture. Noimmunosuppressive drugs were used.

As discussed below, the beads generally reversed hyperglycemia in thehosts. This condition was confirmed by histological analysis. Theencapsulated islets were recovered from streptozotocin-induced diabeticanimals sacrificed two weeks after implantation and were routinely fixedand examined histologically. Donor islets were fixed in Bouin'ssolution, and then dehydrated and embedded in paraffin by routinehistologic methods. The tissue was sectioned serially (5 μm sections)and stained with hematoxylin-eosin. The presence of insulin, glucagon,and somatostatin in donor islets was determined using immunoperoxidasehistochemistry as described in Warnke et al., J. Histochem. Cytochem,28:771 (1980) or Like et al., Lab. Invest.; 38:340 (1978). These testsare used to determine whether all these hormone-secreting cell types inthe islets are viable.

Such histological tests are the only accurate method to determine theviability of islets after implantation in chemically induced diabeticanimals, because it is common for such animals to revert to anon-diabetic state, which gives a false indication of islet viability ifdetermined only by blood tests such as blood glucose levels. Inaddition, blood tests provide no indication of fibrosis.

As shown in FIG. 2, porcine islets (approximately 20,000) encapsulatedin 800±100 μm diameter beads made from 1.0 ml of alginate gel, andimplanted into diabetic mice, reversed hyperglycemia for more than 10weeks as evidenced by a drop in the plasma glucose level immediatelyafter implantation from about 400 to 450 mg/dl to about 200 mg/dl, andthe maintenance of the plasma glucose level at about 200 to 250 mg/dl.

These results were confirmed by histological analysis, whichdemonstrated intact, viable islets after 10 weeks. In addition, thebeads showed little or no fibrosis for the 10 week period. Controlexperiments in which approximately 100,000 non-encapsulation canine,bovine, or porcine islets were implanted intraperitoneally intoSTZ-induced diabetic Lewis rats showed that these xenografts all failedwithin one week.

In other experiments, porcine islets were immobilized in five differentsizes of uncoated alginate beads (880, 1600, 2200, 3000, and 3700 μm indiameter). These were implanted into the peritoneal cavity ofSTZ-induced diabetic rats for 11 to 14 days (n=2 for all tests). Noimmunosuppressant was used in these experiments. No islets survived inany of the 800 μm or 1600 μm diameter beads. Thus, it appears thatsmaller diameter beads containing porcine islets do not work withoutimmunosuppression in rats. In other experiments, loss of blood glucosecontrol and histology confirm that smaller diameter gel beads arerejected within about 6 to 10 days after implantation.

However, discordant xenograft studies using larger beads, e.g., 2200,3000, and 3700 μm diameter beads, showed that porcine islet cellsremained viable for more than four weeks in rats without anyimmunosuppression. The results (percent viability after 4 weeks) areshown in Table 1 below.

                  TABLE 1    ______________________________________    Uncoated Bead Diameter (μm)                       Percent Viability    ______________________________________     800               0    1600               0    2200               33 to 44    3000               50 to 75    3700               80 to 85    ______________________________________

Thus, larger diameter beads are successful in protecting the donorporcine islets from the host rats' immune systems without anyimmunosuppressant or anti-fibrotic drugs.

Implantation of Bovine Pancreatic Islets into Mice and Rats

In another discordant xenograft study, bovine calf pancreatic isletcells were isolated and encapsulated as described above for the porcineislets. Again, these beads were analyzed in the mouse and rat diabetesmellitus model described above.

As shown in FIG. 3, bovine islets (approximately 20,000) encapsulated in800±100 μm diameter beads made from 1.0 ml of alginate gel, andimplanted into mice reversed hyperglycemia for greater than 60 days asevidenced by the immediate drop in plasma glucose level afterimplantation from about 550 mg/dl to about 150 mg/dl, and themaintenance of this level for the duration of the study. Histologicalanalysis demonstrated intact, viable islets for more than 60 days, withlittle or no fibrosis.

Similarly, different numbers of encapsulated bovine islets wereimplanted into rats. CsA was administered subcutaneously to the rats forthe first two weeks of the study and then discontinued (30 mg/kg on days-1, 0, and 1; 15 mg/kg on days 2 to 5; and 7 mg/kg on days 6 to 14). Asshown in FIG. 4, all four dosage levels of islets (60K, 80K, 100K, and120K) reversed hyperglycemia for more than 40 days. Plasma glucoselevels fell from about 525 to 725 mg/dl to less than 250 mg/dl afterimplantation, and maintained these levels for the duration of the study.

Histological analysis demonstrated intact, viable islets at 64 days,with little or no fibrosis.

Implantation of Islet Cells into Dogs

In an allograft study, donor canine islets (approximately 100,000) wereencapsulated in 800±100 μm diameter beads made from 5.0 ml of alginategel and implanted into dog hosts. Although such allografts are normallyrejected by a host within 7 days, the encapsulated donor islets were allviable after three weeks. One dog received no immunosuppressant drug,and another received one daily injection of 10 mg/kg CsA, which providesa blood trough level of about 200 to 300 ng/ml, depending on the actualtime of administration, liver function, and time of measurement. Therewas no fibrosis of the implanted beads in either dog, even without theuse of any immunosuppressive agent in one of the dogs. Histology showedthat 50% of the islets were viable in both dogs after three weeks. Thus,the allografts were successful with or without the use ofimmunosuppressant or anti-fibrotic drugs.

In a discordant xenograft dog study, porcine pancreatic islets(approximately 140,000) were isolated and encapsulated in 800±100 μmbeads made from 7.0 ml of alginate gel and implanted as described abovefor the porcine islets. These beads were analyzed in a normal dog. Theimmunosuppressant CsA was administered orally to the dog at a dosage of10 mg/kg/day throughout the course of the study. Histological analysisshowed some viable islets after three weeks.

As a control, empty beads of the same size were injected into dogs todetermine whether the beads themselves caused any inflammation or otherimmune reaction in the host. Histological examination showed thatneither the empty nor the islet-containing beads had developed anyfibrosis after over one month inside the dog host.

Treatment of Diabetic Dogs

In another dog study, two actual diabetic patient dogs were treated byimplantation of about 600,00 to 650,000 canine islets in 800±100 μmbeads made from 30.0 to 33.0 ml of alginate gel and implanted asdescribed above. The first diabetic dog required about 11 to 12 units ofinsulin per day prior to implantation. As shown in FIG. 5A theseimplanted beads reversed hyperglycemia for over six weeks as evidencedby the immediate reversal of hyperglycemia (drop in plasma glucose levelafter implantation from about 650 mg/dl to about 150 mg/dl), and themaintenance of the plasma glucose level at about 125 mg/dl for theduration of the study, without the need for any external administrationof insulin.

In addition, at one month after implantation, a bolus intravenousinjection of glucose caused only a transient rise in blood glucose levelto 300 mg/dl, which was normalized within about 1 hour (data not shown).Prior to implantation, the same test showed a sugar level of over 600mg/dl and remained significantly hyperglycemic for the duration of thetest (greater than 600). These pre- and post-implantation tests provideevidence that the animals are truly diabetic absent the implants.

The second diabetic dog required about 8 to 10 units of insulin per dayprior to implantation. As shown in FIG. 5B these implanted beadsreversed hyperglycemia for over 7 days as evidenced by the immediatedrop in plasma glucose level after implantation from about 350 mg/dl toabout 100 mg/dl, and the maintenance of the plasma glucose level atabout 100 mg/dl for the duration of the study, without the need for anyexternal administration of insulin.

In this study, a low dose of CsA was administered to each dog. CsA wasadministered at a dosage of 10 mg/kg/day for the first two weeks, andthen dropped to 5 mg/kg/day. However, by 21 days, HPLC analysis of thedog's blood showed no detectable trace of CsA. Thus, the blood level ofCsA was less than about 30 ng/ml, which is the lowest detectable limitof this measurement technique.

Factor IX Expressing Cells

HeLa cells, primary rabbit fibroblast cells (WHHL), and hepatoma cells(HepG2) were engineered to overexpress human Factor IX by transfectionwith one of two human Factor IX expressing retroviral sequences, Moloneymurine Leukemia virus LTR (Mo-LTR) or Myeloproliferative sarcoma virusLTR (MPSV-LTR). Human FIX is expressed from the retroviral 5' LTRpromotor, while the dominant selectable marker npt (neomycinphosphotransferase; neo resistance; G418 resistance) is expressed froman internal promoter. Three of the four cell populations (one of each ofHeLa, WHHL, and HepG2) were generated with the same retroviral vectorwhich uses the Mo-LTR. The fourth population (WHHL) was generated usingMPSV-LTR. All four cell populations secreted detectable levels of humanFIX as determined by ELISA.

These cells were then encapsulated as follows. 1×10⁶ or 2×10⁶ cells wereencapsulated in 800±100 μm diameter beads made from 1.0 ml of alginate.These beads were then cultured in vitro in DMEM in high glucose (G418 ata concentration of 0.8 mg/ml) to determine the amount of human Factor IXsecreted by these encapsulated cells. The encapsulated cells producedhigh levels of Factor IX. Selection for G418 should be applied if thecells are grown for more than 21/2 weeks in culture.

To determine the levels of Factor IX that these encapsulated cellsproduce in vivo, they are injected i.p. into mice. Untreated mice serveas controls. Plasma human Factor IX concentrations are measured atvarious time intervals after implantataion by tail bleedings using anenzyme immunoassay (Asserachrom IX:Ag; American Bioproducts).

Uses of Uncoated Gel Beads

The uncoated gel beads of the invention can be used to treat a varietyof diseases that result from the defective or insufficient production ofa particular enzyme or hormone by the body. In effect, the currentmethods provide a type of replacement therapy. A number ofwell-characterized disorders caused by the loss or malfunction ofspecific cells in the body are amenable to replacement therapy. Forexample, islets of Langerhans can be used for the treatment of diabetes,hepatocytes for hepatic failure, adrenal gland cells for Parkinson'sdisease, cells that produce nerve growth factor (NGF) for Alzheimer'sdisease, cells that produce factors VIII and IX for hemophilia, andendocrine cells for treating disorders resulting from hormonedeficiency, e.g., hypoparathyroidism.

Moreover, by using recombinant DNA methods, so-called "gene therapy," orencapsulating other tissues, it should also be possible to treatpatients suffering from chronic pain, cancer (e.g., hairy cell leukemia,melanoma, and renal carcinoma), AIDS (treated by immunologicalaugmentation), Kaposi's Sarcoma (treated by administration ofinterferon, IL-2, or TNF-α), primary hematologic disorders, patientswith long-lasting aplasia, and patients who are myelosuppressed (treatedby bone marrow transplantation and aggressive chemotherapy). Uncoatedgel beads should also be useful in the treatment of affective disorders,Huntington's Disease, Duchenne's Muscular Dystrophy, epilepsy,infertility, spinal cord injuries, and in wound healing.

Implantation of specific cells can also serve to detoxify or to removedeleterious substances from the circulation. For example, theimplantation of appropriate living cells restores normal physiologicfunction by providing replacement for the diseased cells, tissues, ororgans, e.g., in hepatic encephalopathy (produced by liver disease) oruremia (produced by kidney failure).

In each application, a sufficient number of uncoated beads, containingthe desired living cells, are implanted into the patient, e.g.,surgically or with a syringe. The beads are implanted, e.g.,intraperitoneally, for a systemic effect, or into a particular location,e.g., the brain to treat Parkinson's disease, or the spinal cord totreat spinal cord injuries, for a local effect.

The dose of uncoated beads to be used is determined initially fromresults of in vitro studies. In addition, in vivo results in, e.g.,mice, rats, or dogs will facilitate more accurate assessment of requireddoses, as these tests are generally predictive of efficacy in humanpatients. For example, spontaneous diabetes in dogs is considered to besimilar to type 1 diabetes in man. Soon-Shiong et al., Transplantation,54:769-774 (1992).

The beads are intended to remain in the patient with viable donor cellsfor extended periods of time up to several months or years. However, ifit is determined that the donor cells are no longer viable, e.g., bymonitoring the patient's blood for a certain level of the proteinsecreted by the donor cells, it is a simple task to renew the supply ofbeads in the patient.

Diabetes Mellitus

To treat diabetes, e.g., in a dog or human patient, the implantablebeads preferably encapsulate isolated canine or porcine islets or othercells that produce insulin or insulin-like growth factor 1 (IGF-1).Islets are prepared and encapsulated using procedures described above.Insulin secretory activity of the encapsulated cells or islets isdetermined both in static culture, e.g., expressed per islet volume, andbased on the capability of the islets to respond to gradedconcentrations of glucose. These values are established as describedabove. Once the insulin secretion activity of a particular batch ofencapsulated islets is determined, the proper number of beads can bedetermined and implanted into a diabetic patient. For example, to treata human patient that requires 20 to 50 units of insulin per day, thetotal number of beads should be selected to contain a total of about 1.0to 2.5 million porcine islets. For beads designed to contain, onaverage, 20,000 islets/ml of gel, the proper dosage would be beads madefrom 50 to 125 ml of gel.

Hemophilia

Hemophilia is an X-linked hereditary bleeding disorder caused by FactorVIII or Factor IX deficiency. Recombinant methods have now beensuccessfully used to create Factor VIII- and Factor IX-producing cellsas described above. Encapsulation in uncoated gel beads and implantationof such cells according to the present invention can thus be used for animproved treatment for hemophilia.

Hepatic Diseases

Hepatocyte transplantation is useful not only for irreversible hepaticfailure, but for several disease processes including hereditary enzymeabnormalities, acute hepatic failure, where the ability of the liver toregenerate may still exist, and as a bridge to whole livertransplantation in patients who develop sudden hepatic failure, eitherbecause of medical progression or because of rejection-relatedcomplications.

Wong and Chang, Biomat. Art. Cells Art. Org., 16:731 (1988), havedemonstrated the viability and regeneration of microencapsulated rathepatocytes implanted into mice. Viable hepatocytes weremicroencapsulated in alginate-poly-(L-lysine) and implantedintraperitoneally into normal and galactosamine-induced liver failuremice. Eight days after implantation in the mice with induced liverfailure, the viability of the encapsulated rat hepatocytes increasedfrom 42% to nearly 100%. After 29 days, the viability of theencapsulated hepatocytes implanted in normal mice also increased from42% to nearly 100%. By contrast, free rat hepatocytes implanted intomice all died within four or five days after xenotransplantation. Theuncoated beads of the invention are well-suited to treat hepaticfailure.

Other investigators have shown that microencapsulated hepatocytescontinue the synthesis and secretion of many specific proteins andenzymes. Cai et al., Hepatology, 10:855 (1989), developed and evaluateda system of microencapsulation of primary rat hepatocytes. Ureaformation, prothrombin and cholinesterase activity, the incorporation oftritiated leucine into intracellular proteins, and the immunolocation ofsynthesized albumin were monitored in culture. Despite gradual decreasesin some of these activities, the encapsulated hepatocytes continued tofunction throughout the 35-day observation period. In addition, Bruniand Chang, Biomat. Art. Cells Art. Org., 17:403 (1989),demonstrated theuse of microencapsulated hepatocytes to lower bilirubin levels inhyperbilirubinemia. Microencapsulated hepatocytes were injected into theperitoneal cavity of Grunn rats. Bilirubin dropped from 14mg/100ml to 6mg/100ml, and remained depressed after 90 days. Again, the uncoated gelbeads of the invention can be used as described above to treat thesehepatic diseases.

Parkinson's Disease

Parkinson's disease is a neuronal system disease, involving adegeneration of the nigrostriatal dopaminergic system. Experimental workin both rodents and nonhuman primates has shown that transplantation offetal tissue containing substantia nigra (dopaminergic) neurons fromventral mesencephalon to dopamine-depleted striatum reinstatesnear-normal dopamine innervation and reduces motor abnormalities. Inaddition, implantation of adrenal chromaffin cells has been shown toreverse chemically-induced Parkinson's disease in rodents.

Widner et al., Transplant Proc., 23:793 (1991), recently reportedevidence of fetal nigral allograft survival and function up to 10 monthsafter transplantation and immunosuppression (cyclosporine, azathioprine,and prednisone) in a human Parkinson's patient. Beginning from thesecond month after the transplantation, they observed a progressivedecrease in limb rigidity, increased movement speed in a number of arm,hand, and foot movements, and prolonged "on" periods (>80% increase)after a single dose of L-dopa.

Thus, transplantation of fetal neural tissue, or cells geneticallyengineered to produce dopamine and nerve growth factors or otherneurotropic factors, should have a great potential as a new therapeuticapproach in patients with neurological disorders. However, in the caseof transplanted xenogeneic donor tissue, rejection would pose a seriousproblem, even by the combined approach of using an immunoprivileged siteand by employing immunosuppressive drugs. Therefore the uncoated beadsof the invention permit a novel approach to this problem, i.e., thedelivery of dopamine for the treatment of Parkinson's disease usingencapsulated donor tissue harvested from animals or geneticallyengineered cells.

Alzheimer's Disease

An estimated 2.5 to 3.0 million Americans are afflicted with Alzheimer'sdisease. The disease is characterized by a progressive loss of cognitivefunction associated with degeneration of basal forebrain cholinergicneurons. Studies in animals indicate that Nerve Growth Factor (NGF),e.g., brain-derived neurotrophic factor (BDNF) and neurotrophin-3(NT-3), available from Regeneron and Amgen, respectively, and otherneurotropic factors normally act to support the viability and functionof these neuron cells, and that continuous infusion of NGF into theventricles can prevent injury-induced degeneration of cholinergicneurons as described in Williams et al., P.N.A.S., USA, 83:9231 (1986).This treatment correlates with improved cognitive function in rodentswith memory impairment as described in Fisher et al., Neurobiol. Aging,10:89 (1989).

These studies suggest that uncoated gel beads containing grafts ofrecombinant or natural NGF-secreting tissue such as astroglial cells ordeveloping skin, can be used to treat patients suffering fromAlzheimer's disease.

Gene Therapy

Gene therapy is an approach to treating a broad range of diseases bydelivering therapeutic genes directly into the human body. Diseases thatcan potentially be cured by gene therapy include diseases associatedwith the aging population such as cancer, heart disease, Alzheimer'sdisease, high blood pressure, atherosclerosis and arthritis; viralinfectious diseases such as acquired immune deficiency syndrome (AIDS)and herpes; and inherited diseases such as diabetes, hemophilia, cysticfibrosis, and muscular dystrophy.

In one particular example, a favored approach for human gene therapyinvolves the transplantation of genetically-altered cells into patients,e.g., as described Rosenberg, et al., New Eng. J. Med., 323:570-578(1988). This approach requires the surgical removal of cells from eachpatient to isolate target cells from nontarget cells. Genes areintroduced into these cells via viral vectors or other means, followedby transplantation of the genetically-altered cells back into thepatient. Although this approach is useful for purposes such as enzymereplacement therapy (for example, for transplantation into a patient ofcells that secrete a hormone that diseased cells can no longer secrete),transplantation strategies are less likely to be suitable for treatingdiseases such as cystic fibrosis or cancer, where the diseased cellsthemselves must be corrected. Other problems commonly encountered withthis approach include technical problems, including inefficienttransduction of stem cells, low expression of the transgene, and growthof cells in tissue culture which may select for cells that arepredisposed to cancer.

The uncoated particles of the invention are well suited to avoid theseproblems, because they allow the use of standard human cell lines of,e.g., fibroblast cells, epithelial cells such as HeLa cells, andhepatoma cells such as HepG2, as the implanted cells, rather thanrequiring the surgical removal of cells from the patient. These celllines are genetically altered as required by standard techniques and areencapsulated and implanted into the patient. These cell lines are mucheasier to obtain, culture, and work with than individual patients'cells. Moreover, since the uncoated particles prevent the patient'simmune system from recognizing and attacking the implanted cells, anyhuman cell lines can be used, making the technique of gene therapy moreuniversally applicable.

Hypoparathyrodism

Acute and chronic symptoms of hypoparathyroidism result from untreatedhypocalcemia, and are shared by both hereditary and acquiredhypoparathyroidism. The hereditary form typically occurs as an isolatedentity without other endocrine or dermatologic manifestations or, moretypically, in association with other abnormalities such as defectivedevelopment of the thymus or failure of other endocrine organs such asthe thyroid or ovary. Acquired hypoparathyroidism is usually the resultof inadvertent surgical removal of all the parathyroid glands, and is aproblem in patients undergoing operations secondary to parathyroidadenoma or hyperplasia. Hypoparathyroidism has been treated inhypocalcemic rats by the administration of microencapsulated parathyroidcells that served as a bioartificial parathyroid. Parathyroid cells canalso be encapsulated in the uncoated gel beads of the invention for usein administration to animal and human patients.

Osteoporosis

The term osteoporosis covers diseases of diverse etiology that cause areduction in the mass of bone per unit volume. These diseases can betreated by the administration of uncoated gel beads containing cellsthat secrete insulin-like growth factor (IGF-1), estrogen inpostmenopausal woman to reduce the negative calcium balance and decreaseurinary hydroxyproline, androgens in the treatment of osteoporotic menwith gonadal deficiency, or calcitonin for use in establishedosteoporosis.

Reproductive Disorders

There are numerous disorders of the ovary and female reproductive tractthat can be treated with progestogens, estrogens, and other hormones.These include progestogen, e.g., progesterone, therapy to inhibitpituitary gonadotropins (precocious puberty in girls), and forprophylaxis to prevent hyperplasia in PCOD. Estrogen therapy is used inthe treatment of gonadal failure, control of fertility, and in themanagement of dysfunctional uterine bleeding. Androgens, gonadotropins,and other hormones are used to treat disorders of the testis, e.g.,androgen therapy in hypogonadal men, or gonadotropins to establish orrestore fertility in patients with gonadotropin deficiency. Accordingly,these diseases can be treated with uncoated beads containing theappropriate hormone-producing cells.

Huntington's Disease

Huntington's disease is characterized by a combination ofchoreoathetotic movements and progressive dementia usually beginning inmidadult life. Distinctive for the disease is atrophy of the caudatenucleus and, to a lesser extent, other structures of the basal ganglia(putamen and globus pallidus). Rodent cells that secrete neurotropicfactors have been implanted into the brains of baboons that have acondition similar to Huntington's disease and reversed some of thedamaged nerve networks that, in Huntington's patients, lead toprogressive loss of control over the body. Similarly, Huntington'sdisease in human patients can be treated by the administration ofuncoated beads that contain human or recombinant cells that secrete theappropriate neurotrophic factors.

Spinal Cord Injuries

The majority of spinal cord injuries result from damage to thesurrounding vertebral column, from fracture, dislocation, or both.Treatment of such injuries involves the administration of nerve growthfactors such as ciliary neurotropic factor (CNTF), insulin-like growthfactor (IGF-1), and neurotropic factors, to enhance the repair of thecentral and peripheral nervous system. Thus, uncoated gel beadscontaining cells that secrete such factors, either naturally or throughgenetic engineering, can be used to treat spinal cord injuries.

Mood (or Affective) Disorders

Mood disorders are a group of mental disorders such as schizophreniacharacterized by extreme exaggerations and disturbances of mood andaffect associated with physiologic (vegetative), cognitive, andpsychomotor dysfunctions. Many mood disorders are associated withmedical diseases that can be treated with uncoated gel beads containingthe appropriate cells such as hypothyroidism, Parkinson's disease,Alzheimer's disease, and malignancies as discussed herein. In addition,it has been shown that the neurotransmitter 5-hydroxyindol acetic acid(5-HIAA), a serotonin metabolite, is reduced in the cerebral spinalfluid of depressed patients. Deficits in other neurotransmitters such asdopamine and gamma-aminobutyric acid (GABA) have also been identified inpatients with major depression. Therefore, uncoated gel beads containingcells that secrete these neurotransmitter are useful to treat thesedeficiencies.

Motor Neuron Diseases

Degenerative motor neuron diseases include ALS (see above), heritablemotor neuron diseases (spinal muscular atrophy (SMA), and thoseassociated with other degenerative disorders such asolivopontocerebellar atrophies and peroneal muscular atrophy. Thesediseases can be treated by administration of uncoated gel beadscontaining cells that secrete neurotropic factors like brain-derivedneurotrophic factor (BDNF), and neurotrophin-3 (NT-3).

Acquired Immunodeficiency Syndrome (AIDS)

AIDS is caused by an underlying defect in cell-mediated immunity due tothe human immunodeficiency virus (HIV), and causes persistentconstitutional symptoms and/or diseases such as secondary infections,neoplasms, and neurologic disease. Patients can be treated to amelioratesymptoms by immunologic augmentation with uncoated beads that containcells genetically engineered to secrete, e.g., recombinant human IL-2(to decrease suppressor cell activity resulting in an increased T celladjuvant activity); or recombinant human INF-γ (macrophageaugmentation). AIDS-related tumors such as Kaposi's sarcoma can betreated with encapsulated cells that secrete human interferon-α,interleukin-2 and tumor necrosis factor (TNF).

μmyotrophic Lateral Sclerosis fLou Gehrig's Disease)

ALS is the most frequently encountered form of progressive motor neurondisease, and is characterized by progressive loss of motor neurons, bothin the cerebral cortex and in the anterior horns of the spinal cord,together with their homologs in motor nuclei of the brainstem. ALS canbe treated with uncoated beads that contain cells that secrete nervegrowth factors such as myotrophin, insulin-like growth factor (IGF-1),ciliary neurotropic factor (CNTF), brain-derived neurotrophic factor(BDNF) and neurotrophin-3 (NT-3). Animal studies with these factors(IGF-1 is available from Cephalon, CNTF from Regeneton, and NT-3 fromAmgen), have demonstrated that they can stem the degenerative effectscaused by nerve damage or disease.

Cancer

In most cases, cancer originates from a single stem cell whichproliferates to form a clone of malignant cells. Growth is not properlyregulated by the normal biochemical and physical influences in theenvironment. There is also a lack of normal, coordinated celldifferentiation. Cancer cells develop the capacity for discontinuousgrowth and dissemination to other parts of the body.

Various cancers can be treated according to the invention by theadministration of uncoated gel beads containing cells that secreteinterferon-α (IFN-α) (for solid tumors, hairy cell leukemia, Kaposi'ssarcoma, osteosarcoma, and various lymphomas); recombinant interleukin-2(IL-2) (for melanoma, renal carcinoma, and Kaposi's sarcoma); tumornecrosis factor (w/IL-2 for Kaposi's sarcoma); recombinant human IFN-αand recombinant human colony stimulating factor-granulocyte macrophage(CSF-gm) (for Kaposi's sarcoma); recombinant human INF-γ (for macrophageaugmentation); CSF (for aggressive chemotherapy, bone marrowtransplantation, priming of leukemic cells to enhance sensitivity tochemotherapy and to support dose intensification); ciliary neurotropicfactor (CNTF) and insulin-like growth factor (IGF-1) (for peripheralneuropathies caused by chemotherapy); adrenal gland cells (for painrelief when injected into the lower spine to secrete naturalpainkillers) and progestogen-producing cells (for palliation inendometrial and breast carcinoma).

Duchenne's Muscular Dystrophy

Duchenne's dystrophy is an X-linked recessive disorder characterized byprogressive weakness of girdle muscles, inability to walk after age 12,kyphoscoliosis (curvature of the spine), and respiratory failure afterthe fourth decade. This disease can be treated by administration ofuncoated beads containing myoblast cells and growth factors. Myoblastshave been injected into young boys with Duchenne's muscular dystrophy todetermine whether the cells can supply a structural protein that ismissing. Researchers have observed muscle strength improvement inseveral of the boys.

Epilepsy

The epilepsies are a group of disorders characterized by chronic,recurrent, paroxysmal changes in neurologic function caused byabnormalities in the electrical activity of the brain. In some forms offocal epilepsy, inhibitory interneurons appear to be preferentiallylost. Treatment with neurotropic factors and other neuropeptides such ashas been found effective. Therefore, the uncoated beads of the inventioncontaining cells secreting these factors can be used to treat epilepsy.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

What is claimed is:
 1. A method of implanting a living donor cell into ahost animal, wherein said animal is a mammal larger than a rat or mouse,with minimal inflammatory response or rejection of the donor cell by thehost animal, said method comprising the steps ofobtaining an uncoatedparticle consisting essentially of a biocompatible,temperature-independent gel that encapsulates the living donor cell,wherein said uncoated particle provides a molecular weight cutoff thatprevents host animal immune cells from entering the particle, and doesnot prevent entry of host animal IgG and complement into said particle,wherein said particle has a diameter of 700-6,000 microns, andimplanting the uncoated particle into the host animal.
 2. A method ofclaim 1, wherein said donor cell is obtained from an species that is thesame as the host animal.
 3. A method of claim 2, wherein the host animalis a dog.
 4. A method of claim 2, wherein the host animal is a human. 5.A method of claim 4, wherein the donor cell is a genetically alteredhuman cell.
 6. A method of claim 1, wherein the donor cell is obtainedfrom an animal species that is different from the host animal.
 7. Amethod of claim 6, wherein the host animal is a dog.
 8. A method ofclaim 6, wherein the host animal is a human.
 9. A method of claim 8,wherein the donor cell is a porcine, bovine, or canine cell.
 10. Amethod of claim 8, wherein the donor cell is a pancreatic islet cell.11. A method of claim 1; wherein the donor cell secretes Factor IX,Factor VIII, an interleukin, an interferon, or an endocrine hormone. 12.A method of claim 1, wherein the donor cell secretes a nerve growthfactor, tumor necrosis factor alpha, a neurotropic factor, or aneurotransmitter.
 13. A method of claim 1, wherein said gel particle isspherical and has a diameter of from 1,500-3,500 microns.
 14. A methodof claim 13, wherein said gel particle has a diameter of from 2000 to4500 microns.
 15. A method of claim 1, wherein said gel is an alginateor alginate derivative.
 16. A method of claim 15, wherein said alginateis crosslinked with an ion.
 17. A method of claim 16, wherein saidalginate is crosslinked with a calcium salt.
 18. A method of claim 1,wherein said uncoated gel particle is biodegradable.
 19. A method ofclaim 18, wherein a rate of degradation of said gel in said uncoatedparticle is selected to match a life expectancy of said donor cell. 20.A method of claim 1, wherein said uncoated particle encapsulates anautologous erythrocyte in addition to the donor cell.
 21. A method ofclaim 1, wherein said uncoated particle containing a living cell istreated with a nitric oxide inhibitor prior to implantation.
 22. Amethod of claim 1, further comprising the step of administering a drugto the host animal at a dosage effective to inhibit fibrosis andinflammation around said uncoated particle, but at a dosage lower thanthat required to achieve immunosuppression when said donor cell isimplanted into the host animal without encapsulation.
 23. A method ofclaim 22, wherein said drug is cyclosporin A and is administered at adosage that achieves a whole blood trough level of less than about 100ng/ml in the host animal.
 24. A method of claim 22, wherein said drug isadministered for up to one month after implantation, and is then nolonger administered.
 25. A method of implanting a living donor cell intoa host animal, wherein said animal is a mammal larger than a rat ormouse, with inflammatory response or rejection of the donor cell by thehost animal, said method comprising the steps ofsuspending the livingdonor cell in a liquid medium, said medium consisting essentially ofwater and a biocompatible, temperature-independent liquid gel, forming adroplet of said liquid medium that contains at least one living cell,solidifying said droplet to form a gel particle that encapsulates theliving cell, whereby no outer coating is formed on said particle, andwherein said uncoated particle provides a molecular weight cutoff thatprevents host animal immune cells from entering the particle, and doesnot prevent entry of host animal IgG and complement into said particle,wherein said particle has a diameter of 700-6,000 microns, andimplanting the uncoated particle into the host.
 26. A method of claim25, wherein said liquid medium contains pancreatic islets.
 27. A methodof claim 25, wherein said pancreatic islets are contained in said liquidmedium at a density of 10,000 to 35,000 islets per milliliter of saidmedium.
 28. A method of claim 25, wherein said liquid medium containsliving cells at a density of about 10⁵ to 10⁸ cells per milliliter ofsaid medium.
 29. A method of treating a disease in a patient, whereinsaid patient is a mammal larger than a rat or mouse, caused by adeficient production of a substance in the patient, said methodcomprising the steps ofobtaining an uncoated particle consistingessentially of a biocompatible, temperature- independent gel thatencapsulates a living donor cell that secretes said substance, whereinsaid uncoated particle provides a molecular weight cutoff that preventspatient immune cells from entering the particle, and does not prevententry of patient IgG and complement into said particle, wherein saidparticle has a diameter of 700-6,000 microns, and implanting theuncoated particle into the patient in a location and in a manner thatallows the living cell to remain physiologically active and secrete saidsubstance into the patient to treat said disease.
 30. A method of claim29, wherein the donor cell is obtained from an animal species that isthe same as the patient.
 31. A method of claim 30, wherein the patientis a dog.
 32. A method of claim 30, wherein the patient is a human. 33.A method of claim 32, wherein the donor cell is a genetically alteredhuman cell.
 34. A method of claim 29, wherein the donor cell is obtainedfrom a species that is different from the patient.
 35. A method of claim34, wherein the patient is a dog.
 36. A method of claim 34, wherein thepatient is a human.
 37. A method of claim 29, wherein the donor cell isa porcine, bovine, canine, bacterial, fungal, or plant cell.
 38. Amethod of claim 29, wherein the disease is diabetes and the donor cellis a pancreatic islet cell.
 39. A method of claim 29, wherein the donorcell secretes Factor IX, Factor VIII, an interleukin, an interferon, anendocrine hormone, a nerve growth factor, tumor necrosis factor alpha, aneurotropic factor, or a neurotransmitter.
 40. A method of claim 29,wherein the disease is diabetes mellitus, hepatic disease, amyotrophiclateral sclerosis, hemophilia, hypothyroidism, Parkinson's disease,acquired immune deficiency syndrome, Duchenne's muscular dystrophy,infertility, epilepsy, Huntington's disease, hypoparathyroidism, a mooddisorder, a motor neuron disease, osteoporosis, or Alzheimer's disease.41. A method of claim 29, wherein the gel particles are implanted intoan immunoprivileged site in the patient.
 42. An in vivo method ofculturing a living cell, said method comprising the stepsofencapsulating the living cell in an uncoated particle consistingessentially of a biocompatible, temperature-independent gel, insertingsaid uncoated particle into an animal, and allowing said animal tothrive, thereby culturing the cell wherein said animal is a mammallarger than a rat or mouse, wherein said particle has a diameter of700-6,000 microns and wherein said uncoated particle provides amolecular weight cutoff that prevents host animal immune cells fromentering the particle, and does not prevent entry of host animal IgG andcomplement into said particle.
 43. An in vitro method of culturing aliving cell, said method comprising the steps ofencapsulating the livingcell in an uncoated particle consisting essentially of a biocompatible,temperature-independent gel, wherein said particle has a diameter of700-6,000 microns and wherein said uncoated particle provides amolecular weight cutoff that prevents host animal immune cells fromentering the particle, and does not prevent entry of host animal IgG andcomplement into said particle placing said uncoated particle into amedium including nutrients and oxygen, and maintaining a sufficientamount of nutrients and oxygen in said medium to allow the cell tothrive, thereby culturing the cell.
 44. A method of manufacturinguncoated, temperature-independent gel particles containing living cellsconsisting of the steps ofsuspending the living cells in a liquidmedium, said medium consisting essentially of water and a biocompatible,temperature-independent, liquid gel, forming a droplet of said liquidmedium, solidifying said droplet to form a gel particle thatencapsulates the living cells, whereby no outer coating is formed onsaid particle, wherein said particle has a diameter of 700-6,000 μm andwherein said uncoated particle provides a molecular weight cutoff thatprevents host animal immune cells from entering the particle, and doesnot prevent entry of host animal IgG and complement into said particle,and storing said gelled uncoated particles in a nutrient medium tomaintain the viability of the living cells.
 45. A method of claim 22,wherein said drug is a nonsteroidal anti-inflammatory drug.
 46. A methodof implanting a living donor cell into a host animal, wherein saidanimal is a mammal larger than a rat or mouse, with minimal inflammatoryresponse or rejection of the donor cell by the host animal, said methodcomprising the steps ofobtaining an uncoated particle consistingessentially of a biocompatible, temperature- independent gel thatencapsulates the living donor cell, wherein said uncoated particleprovides a molecular weight cutoff that prevents host animal immunecells from entering the particle, and does not prevent entry of hostanimal IgG and complement into said particle, implanting the uncoatedparticle into the host animal, and administering a drug to the hostanimal at a dosage effective to inhibit fibrosis and inflammation of theuncoated particle, but at a dosage lower than that required to achieveimmunosuppression when the donor cell is implanted into the host animalwithout encapsulation.
 47. A method of claim 46, wherein said donor cellis obtained from an species that is the same as the host animal.
 48. Amethod of claim 46, wherein the host animal is a human.
 49. A method ofclaim 46, wherein the donor cell is a genetically altered human cell.50. A method of claim 46, wherein the donor cell is obtained from ananimal species that is different from the host animal.
 51. A method ofclaim 50, wherein the host animal is a human.
 52. A method of claim 50,wherein the donor cell is a porcine, bovine, or canine cell.
 53. Amethod of claim 46, wherein the donor cell is a pancreatic islet cell.54. A method of claim 46, wherein the donor cell secretes Factor IX,Factor VIII, an interleukin, an interferon, or an endocrine hormone. 55.A method of claim 46, wherein the donor cell secretes a nerve growthfactor, tumor necrosis factor alpha, a neurotropic factor, or aneurotransmitter.
 56. A method of claim 46, wherein said gel particle isspherical and has a diameter of from 50 to 6000 microns.
 57. A method ofclaim 46, wherein said gel is an alginate or alginate derivative.
 58. Amethod of claim 46, wherein said drug is cyclosporin A and isadministered at a dosage that achieves a whole blood trough level ofless than about 100 ng/ml in the host animal.
 59. A method of claim 46,wherein said drug is administered for up to one month afterimplantation, and is then no longer administered.
 60. A method of claim46, wherein said drug is a nonsteroidal anti-inflammatory drug.
 61. Amethod of claim 1, wherein said particle is other than a sphere.
 62. Amethod of claim 25, wherein said particle is other than a sphere.
 63. Amethod of claim 29, wherein said particle is other than a sphere.
 64. Amethod of claim 46, wherein said particle is other than a sphere.